The goals of this project are to understand the nature of kinetic barriers in protein folding, and to determine the roles that partially structured intermediates play in this reaction. These frequently-observed species lie at the center of the controversy that currently divides hypotheses for how proteins fold. One view regards folding as a process that proceeds through discrete intermediates in a well-defined pathway. The other considers folding as a continuum of states and pathways, with intermediates being optional pits in the energy landscape. To test these hypotheses, a novel method has been developed for discriminating protein sequences based on the speeds at which they fold. This this kinetic selection will be used to isolate fast- and slow-folding variants from combinatorial libraries of apomyoglobin mutants. The types, locations, and structural contexts of the mutations that give rise to fast and slow folding will identify the rate-limiting interactions in apomyoglobin folding. Individual fast- and slow-folding mutants will be constructed. Stop-flow fluorescence experiments will elucidate their kinetic folding mechanisms. Changes in backbone hydrogen bonding during folding will be characterized by pulsed amide hydrogen exchange studies. Structures of mutant proteins in their native and intermediate states will be investigated by nuclear magnetic resonance experiments. These combined studies will test the two hypotheses by determining if fast- and slow-folding of mutants share a common folding mechanism, or if they fold by altogether different routes. This proposal develops new approaches that address the following interrelated questions. (i) Do intermediates speed up or slow down folding? (ii) What is the nature of the rate-limiting step? (iii) Are kinetic barriers optional or intrinsic? (iv) What are the sequence determinants of folding speed? (v) Can we engineer the property of fast folding into proteins? By combining mechanistic experiments with modern combinatorial tools, we hope to delimit the structures and properties of folding intermediates and the energy barriers that link them, which together define the mechanism of protein folding.